The proposed research has three major goals. First (AIM 1); develop genetically modified pigs (GMPs) that overexpress or under-express endothelial nitric oxide synthase (eNOS). We will develop pigs that over-express eNO by adding a construct (composed of an endothelial-specific Tie 2-promoter, full length eNOS cDNA and a selectable marker) to Yucatan fetal fibroblast cells and create the pigs using nuclear transfer technology. Such a transgenic animal will over express eNOS specifically in endothelial cells. We will develop pigs that lack eNOS (eNOS-/-) by replacin exon 12 with a neomycin cassette by homologous recombination in Yucatan fetal fibroblast cells. We will produce heterozygous eNOS (+/-) pigs using targeted fibroblast cells for nuclear transfer, eNOS-/- pigs, that lack functional eNOS protein in all cell types, will be generated from the breeding of eNOS pigs. Second (AIM 2); establish the metabolic and cardiorespiratory phenotype of these GMPs. We will characterize whole body metabolism ana cardiorespiratory functional capacities of GMPs and the regulation of coronary blood flow. From the same animals we will also characterize the effects of altered eNOS expression on metabolism of skeletal muscle and fat tissues, ana thephenotype of vascular endothelium and smooth muscle. Finally (AIM 3); determine the effects of altered eNOS expression on endothelial dysfunction and vascular disease produced by high fat diet-induced hyperlipidemia. Pigs share with humans important characteristics of muscle and intermediary metabolism and of cardiorespiratory function, making them another important link in our growing understanding of these processes. These animal models will be powerful tools for investigators at both public and private sector institutions and will form an important tool for our ongoing research concerning the role of eNOS in atherosclerotic vascular disease (supported by NHLBI). In addition, these GMPs will provide important animal models to determine the role of eNOS in: 1) the control of metabolic processes in muscle and fat tissue (NIAMS), 2) diabetes and diabetic vascular disease (NIDDK), and 3) reproductive biology (NICHD). Further development of porcine models for biomedical research will reduce the growing pressure Ion the use of nonhuman primate models for research involving genetic modifications of metabolic and cardiorespiratory systems. The strong group of investigators submitting this application is uniquely qualified and prepared to accomplish these goals.